Research in the Molecular Pathogenesis Section is focused on defining changes in the genes that underlie inherited susceptibilities to common diseases such as cancer and birth defects. Changes in folate and vitamin B12 metabolism are associated with tumor formation, birth defects and cognitive decline. Folate and vitamin B12 genes are also involved in the methylation of DNA and normal brain function. We are searching for genetic variants in genes related to folate, methionine and homocysteine metabolism. Individuals affected with spina bifida (one form of neural tube defects) will be tested for these variants. Variants found at higher frequency in individuals with disease will help us identify genes associated with risk. In the past we found that variants in two genes, TCN2 and TCN2R, appear to affect the levels of vitamin B12 in the blood during pregnancy. In addition, variants in TCN2R are associated the risk of neural tube defects. These findings may be related to birth defects and also may help to explain why some elderly individuals become anemic and suffer neurological symptoms from vitamin B12 deficiency. We also found that mothers carrying a specific variant in a second gene, MTHFD1, have a 50% increased risk bearing a child with a neural tube defect. This previously un-described variant may be responsible for up to 25% of all neural tube defects. Approximately one in five individuals in the population carry one of these risk factors. Over the past year we have carried out series of experiments to use a genome wide association approach to find additional genes that are associated with the risk of bearing a child with a neural tube defect or being an affected individual. The next logical step in this research is to screen the entire genome for additional genes associated with NTDs. This type of experiment requires a very large sample size. Although we have one of the worlds largest samples of NTDs that are available for genetic research, our sample is too small to carry out a genome wide association study (GWAS). In collaboration with Anne Molloy, Trinity College Dublin, we have organized and international collaboration with the goal of pooling samples for a GWAS. Over ten groups have joined this collaborative effort. The total number of samples collected by all groups exceeds 3,000. We have obtained external funding to coordinate this study and collect the samples at a central location. In the past year we have collected DNA samples from these collaborators. These samples have been arrayed into a format that can be used to carry out a GWAS. Notable additions to this collection were samples acquired from the New York State Birth Defects Registry and the CDC sponsored program, the National Birth Defects Prevention Study. These two programs have contributed approximately 600 more cases to this effort. The CDC contributed samples also include the parents of the cases. While the samples from New York are population-based case samples with matched controls. This will allow us to continue to combine family-based and association-based tests. The major challenge in the coming year is to secure funding to carry out this study (the cost exceeds the lab budget). We have continued to work on the biology of the genes we have found to be associated with NTDs. One of these is a gene that produces a protein that binds vitamin B12 and transports it from the blood into the tissues. We have published data demonstrating that several variants in this transporter are associated with a risk of having a child with an NTD. While we now know which variants are associated with risk, we do not yet know if they are actually causing the risk or are linked to additional variants that change the function of the protein. To screen for additional variants, we sequenced the DNA containing this transporter gene in a large number of individuals. This sequencing experiment uncovered a number of previously unidentified variants in this gene. We measured the impact of these variants on the function of the transporter. The majority of variants tested do not appear to have an adverse effect of the function of the receptor. However, we have found that several of the variants are associated with changes in vitamin B12 levels in a large sample of healthy individuals. We tested these variants in a large sample of elderly individuals to determine if specific variants are associated with changes in vitamin B12 levels and disease conditions. We have confirmed that these variants strongly influence vitamin B12 levels in plasma. We then compared the gene variants to disease outcomes in the elderly population. At this time, the variants were not associated with specific diseases in this population. Other groups have measured the impact of genetic variants on the level of vitamin B12 in blood. We have measured vitamin B12 in the blood of over 5,000 individuals using the standard methods. We then subjected these samples to assays that resolve vitamin B12 into pools that correspond to the two major carrier proteins. These measures reveal that different genes influence the levels of the different pools of the vitamin. These findings could be relevant to clinical measures of vitamin B12 testing where it is known that individuals on either side of the normal range are given false positive and negative results. It may be possible to use individual genetic variation to refine the interpretation of clinical testing. We are also using animal models to understand the biology of genes involved in vitamin B12 metabolism. We have developed strains of zebrafish and mice in which we have disrupted vitamin B12 transport genes. These animals appear to mimic a number of aspects of vitamin B12 deficiency in humans. We are in the process of characterizing the biochemical and developmental phenotypes in these animals.